Compositions and methods for detecting cancer metastasis

ABSTRACT

The present invention encompasses compositions and methods for detecting cancer metastasis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Ser. No. 13/243,572, filedSep. 23, 2011, which claims the priority of U.S. provisional applicationNo. 61/385,696, filed Sep. 23, 2010, each of which is herebyincorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under R01 CA125970awarded by the National Cancer Institute, and under P30 EY02687c andAR007279-31A1 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

A paper copy of the sequence listing and a computer readable form of thesame sequence listing are appended below and herein incorporated byreference. The information recorded in computer readable form isidentical to the written sequence listing, according to 37 C.F.R. 1.821(f).

FIELD OF THE INVENTION

The invention encompasses compositions and methods for detecting cancermetastasis.

BACKGROUND OF THE INVENTION

Once a primary tumor has metastasized and is clinically detectable bycurrent diagnostic measures, treatment of the tumor becomes morecomplicated, and generally speaking, survival rates decrease.Consequently, it is advantageous to determine which tumors are morelikely to metastasize and to advance the time to detection ofmetastasis, so that appropriate treatment may be started as soon aspossible. Many different types of tumors are capable of metastasizing.Melanomas, in particular, are capable of aggressive metastasis.

Melanoma is a malignant tumor of melanocytes, and may occur in the eye(uveal melanoma), on the skin, or on mucosal tissues. Uveal melanoma isthe most common intraocular malignancy. The incidence of this tumorincreases with age and reaches a maximum between the 6^(th) and 7^(th)decade of life. Approximately 50% of patients die of metastases, aproportion that, despite all efforts to improve treatment, has remainedconstant during the last century. The average life expectancy afterdiagnosis of metastases is 7 months.

Around 160,000 new cases of melanoma of the skin are diagnosed worldwideeach year, and according to the WHO Report about 48,000 melanoma relateddeaths occur worldwide per annum, which accounts for 75 percent of alldeaths associated with skin cancer. Similar to uveal melanoma, whenthere is distant metastasis, the cancer is generally consideredincurable. The five-year survival rate is less than 10%, with a mediansurvival time of 6 to 12 months. Additionally, specific to uvealmelanoma and cutaneous melanoma and generally considered for carcinoma,earlier treatment of malignancies is associated with improvedprogression-free and overall survival.

Due to the aggressive nature of these malignancies, there is a need inthe art for methods of predicting the risk of metastasis and for earlierdetection of metastatic disease, so that treatment may begin as early aspossible.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a method for determiningthe risk of metastasis in a subject. Generally speaking, the methodcomprises collecting a sample from a subject, analyzing the BAP1nucleotide and/or BAP1 amino acid sequence from a cell in the sample,and identifying the presence of a mutation in the BAP1 nucleotide and/orBAP1 amino acid sequence. The presence of the mutation indicates anincreased risk for metastasis in the subject.

Another aspect of the invention encompasses a method for determining therisk of metastasis in a subject, where the method comprises determiningthe level of BAP1 activity in a sample from a subject, wherein indecreased BAP1 activity indicates an increased risk for metastasis inthe subject.

Still another aspect of the present invention encompasses a method fordetecting the presence of metastatic cancer. Generally speaking, themethod comprises collecting a sample from a subject, analyzing the BAP1nucleotide and/or BAP1 amino acid sequence in the sample, anddetermining the presence of a mutation in the BAP1 nucleotide and/orBAP1 amino acid sequence. The presence of the mutation indicates thepresence of metastatic melanoma.

Yet another aspect of the present invention encompasses a method fordetecting the presence of metastatic cancer, the method comprisingdetermining the level of BAP1 activity in a sample from a subject,wherein decreased BAP1 activity indicates the presence of metastaticcancer in the subject.

Still yet another aspect of the present invention encompasses a methodfor detecting the presence of a biomarker for metastatic cancer in asubject. The method may encompass a method for determining the risk ofmetastasis in a subject. Generally speaking, the method comprisesanalyzing the BAP1 gene nucleotide sequence and/or the BAP1 proteinamino acid sequence from a tumor cell in a sample obtained from thesubject, and identifying the presence of a mutation in the BAP1nucleotide sequence and/or BAP1 protein sequence. The presence orabsence of a mutation is as compared to the gene and/or protein sequencefrom a non-tumor cell from the same subject. For example, the genenucleotide and/or protein amino acid sequence from a non-tumor cell maybe SEQ ID NOs: 3 and 1, respectively. Comparison may also be madebetween cDNA obtained from mRNA from a tumor cell and cDNA obtained frommRNA from a non-tumor cell, which may have BAP1 nucleotide sequence SEQID NO: 2. The presence of a mutation, particularly an inactivatingmutation as defined elsewhere herein, indicates an increased risk formetastasis in the subject.

The biomarker may be decreased BAP1 activity in a tumor cell from asubject, as compared to the activity in a non-tumor cell from the samesubject. Decreased BAP1 activity may be indicative of an increased riskof metastasis in the subject and/or of the presence of metastaticcancer.

Certain aspects of the present invention encompass a method fordetecting the presence of metastatic cancer. Generally speaking, theassay comprises analyzing the BAP1 gene nucleotide sequence or the BAP1protein amino acid sequence in a tumor sample obtained from the subject,and detecting the presence of a mutation in the BAP1 nucleotide sequenceor BAP1 protein sequence, as compared to the sequence in a non-tumorsample from the subject, as mentioned above. The presence of themutation indicates the presence of metastatic melanoma.

Several aspects of the present invention encompasses a metastatic cancerbiomarker, which may be detected in a tumor sample obtained from asubject. The biomarker typically comprises a BAP1 nucleotide sequencecomprising at least one mutation, as compared to the BAP1 sequence in anon-tumor sample from the subject. The biomarker may also comprise aBAP1 amino acid sequence comprising at least one mutation. Such abiomarker may be detectable, for example, by use of an antibody whichspecifically recognizes the biomarker and such antibodies are alsoencompassed by the present invention. The biomarker may be detected bydetecting reduced BAP1 activity in a cell from tumor sample from asubject, as compared to the activity in a cell from a non-tumor samplefrom the same subject.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D illustrate that inactivatingmutations in BAP1 occur frequently in uveal melanomas. (FIG. 1A) Sangersequence traces of MM 056 and MM 070 at the sites of the mutations.Location of mutated base in MM 056 and the start of the deletion of MM070 are indicated (arrows). The non-coding BAP1 strand is shown for MM070. (SEQ ID NOs: 44-47) (FIG. 1B) Map of BAP1 gene and location of BAP1mutations. BAP1 contains 17 exons (shaded boxes) that encode a 728 aminoacid protein. Introns are not to scale. Mutations are shown below thegene figure as indicated. The UCH domain (aa. 1-188) and UCH37-likedomain (ULD) (aa. 635-693) are indicated (12, 13). The critical Q, C, Hand D residues of the active site (Gln85, Cys91, His169 and Asp184) areindicated with asterisks. The catalytic cysteine is indicated with acircle. Also shown are: the NHNY consensus sequence for interaction withHCFC1 (aa. 363-365, exon 11), nuclear localization signals (NLS) at aa.656-661 (exon 15) and aa. 717-722 (exon 17), the BARD1 binding domainwithin the region bounded by aa. 182-240 (13), and the BRCA1 bindingdomain within aa. 598-729 (11). (FIG. 1C) Location of BAP1 gene missensemutations in the UCH domain aligned to the crystal structure of UCH-L3(21). Three-dimensional structure of UCH-L3 was visualized with MMDBsoftware (22). The small molecule near C91W, H169Q and S172R representsa suicide inhibitor, illustrating the critical location of thesemutations for catalytic activity. (FIG. 1D) Conservation of BAP1 inregions containing mutated amino acids. Alignments of segments of BAP1homologs harboring mutated amino acids (missense or in-frame deletions)are shown for the indicated species. (SEQ ID NO: 48-60) Amino acidnumbering is on the basis of human BAP1 (SEQ ID NO: 1). Positions ofmutated amino acids are indicated with asterisks.

FIG. 2 depicts Sanger sequence trace of one end of the mutated region ofNB101. The breakpoint at one end of the insertion/deletion is indicatedwith an arrow. Wild type sequence is indicated below the NB101 sequence.(SEQ ID NO: 61-62)

FIG. 3A and FIG. 3B depict bar graphs of BAP1 mRNA levels. (FIG. 3A) isa graph of BAP1 mRNA levels measured by quantitative RT-PCR in 9non-metastasizing class 1 UMs and 28 metastasizing class 2 Ums, and(FIG. 3B) is a graph showing the relationship between BAP1 mRNA levels(measured by quantitative RT-PCR) and type of BAP1 mutation in 9 UMswith nonsense mutations, 10 UMs with missense mutations (including smallin-frame deletions, splice acceptor, and stop codon read-throughmutations), and 4 class 2 UMs in which no BAP1 mutations were detected.

FIG. 4 depicts a series of photographs illustrating that BAP1 mutationsdisrupt BAP1 protein expression in human uveal melanoma samples.Immunofluorescence analysis of BAP1 protein expression was performed onarchival tumor specimens from uveal melanomas of known class and BAP1mutation status, as indicated. All images were captured at 40× and arerepresented at the same magnification. Scale bar, 10 microns. No BAP1expression is seen in the Class 2 metastasizing UM cells (MM100, MM071,MM135, MM091) whereas expression is seen in the class 1non-metastasizing UM cells (MM050, MM085).

FIG. 5 depicts a series of micrographs illustrating that UM cellsdepleted of BAP1 acquire properties that are typical of metastasizingclass 2 tumor cells. Phase contrast photomicrographs of 92.1 uvealmelanoma cells transfected with BAP1 or control siRNA at the indicateddays. Bottom panels show representative examples of class 1 and class 2uveal melanoma cells obtained from patient biopsy samples (Papanicolaoustain). Scale bars, 10 microns.

FIG. 6 depicts a gene expression heatmap of the top class 1 versus class2 discriminating transcripts in 92.1 uveal melanoma cells transfectedwith control versus BAP1 siRNAs.

FIG. 7A, FIG. 7B and FIG. 7C depict a diagram, a Western blot, and a bargraph, respectively, showing the effects of BAP1 depletion by siRNA.92.1 cells transfected with BAP1 siRNA and evaluated after five days.(FIG. 7A) BAP1 protein levels were efficiently depleted to less than 95%of control levels (see Western blot). Upper panel depicts principalcomponent analysis to show effect of BAP1 knockdown on gene expressionsignature. The small spheres represent the training set of known class 1(blue) and class 2 (red) tumors. Large spheres represent thecontrol-transfected (gray) and BAP1 siRNA transfected (red) cells. Lowerpanel depicts mRNA levels measured by quantitative RT-PCR of a panel ofmelanocyte lineage genes, presented as fold change in BAP1 siRNA/controlsiRNA transfected cells. Results are representative of three independentexperiments. (FIG. 7B) mRNA levels of mRNAs of a panel of melanocytelineage genes measured by quantitative RT-PCR, presented as fold changein BAP1 siRNA/control siRNA transfected cells. (FIG. 7C) RNAi mediateddepletion of BAP1 in 92.1 and Mel290 UM cell lines using two independentsiRNAs that target BAP1. Duplicate experiments of each cell line andsiRNA are shown.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D depict a bar plot (FIG. 8A), aWestern blot (FIG. 8B), and two micrographs, respectively, showing BAP1levels in shGFP (FIG. 8C) and shBAP1 (FIG. 8D) cells.

FIG. 9A and FIG. 9B depict Western blots showing increasedubiquitination of histone H2A in siBAP1 (FIG. 9A) and shBAP1 (FIG. 9B)cells compared to controls. FIG. 9C and FIG. 9D depict fluorescenceimmunohistochemical micrographs showing increased ubiquitination ofhistone H2A in shBAP1 (FIG. 9D) cells compared to control cells (FIG.9C).

FIG. 10A and FIG. 10B depict bar plots from two experiments showingdecreased RNA levels of melanocyte differentiation genes in BAP1 stableknockdown cells.

FIG. 11A, FIG. 11B and FIG. 11C depict plots showing that transientknockdown of BAP1 using siRNA (FIG. 11A) leads to a decrease in cellproliferation. Transient knockdown of BAP1 using shRNA did not altercell proliferation (FIG. 11B and FIG. 11C).

FIG. 12A and FIG. 12B depict micrographs (FIG. 12A) and a bar plot (FIG.12B) showing that loss of BAP1 in culture leads to decreased cellmotility.

FIG. 13A, FIG. 13B and FIG. 13C depict images of shGFP (FIG. 13A) orshBAP1 (FIG. 13B) culture plates and a bar plot (FIG. 13C) showing thatloss of BAP1 leads to decreased growth in soft agar.

FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14 D depict bar plots from fourexperiments showing that loss of BAP1 leads to an increased ability togrow in clonegenic assays.

FIG. 15 depicts a bar plot showing that loss of BAP1 leads to increasedmigration towards a serum attractant.

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E and FIG. 16F depictplots showing that loss of BAP1 in culture leads to decreased tumorgrowth in the mouse flank. FIG. 16A and FIG. 16D depict a decrease inweight of the tumor with loss of BAP1. FIG. 16B and FIG. 16E depict adecrease in volume of the tumor with loss of BAP1. FIG. 16C and FIG. 16Fdepict a decrease of BAP1 RNA expression in the presence of BAP1 shRNA.

FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D depict plots showing that lossof BAP1 in culture leads to decreased tumor growth in the mouse aftertail vein injection.

FIG. 18 depicts an illustration of a family with germline BAP1mutations.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E and FIG. 19F depict thegenomic sequence of BAP1. Exons are bolded, and select mutations arehighlighted (see Table 2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining the risk oftumor metastasis in a subject. Additionally, the invention provides amethod for detecting the presence of a tumor metastasis in a subject.The invention further provides a method for detection of a metastaticcancer biomarker in a subject, wherein detection of the biomarkercomprises identifying a mutation in a BAP1 nucleotide sequence,identifying a mutation in a BAP1 protein sequence, or identifying adecrease in BAP1 activity in a sample obtained from the subject.Advantageously, such methods may allow a physician to determine theseverity of an oncogenic disease in a subject and to make appropriate,timely, treatment decisions based on this information.

I. Method for Determining the Risk of Tumor Metastasis

One aspect of the present invention encompasses a method for determiningthe risk of tumor metastasis in a subject. In one embodiment, the methodcomprises collecting a sample from a subject, analyzing the BAP1nucleotide and/or BAP1 amino acid sequence from a cell in the sample,and identifying the presence of a mutation in the BAP1 nucleotidesequence and/or the BAP1 amino acid sequence. In this context, “amutation in the BAP1 nucleotide sequence,” refers to a mutation in anexon of BAP1, an intron of BAP1, the promoter of BAP1, the 5′untranslated region of BAP1, the 3′ untranslated region of BAP1, or anyother regulatory region for the BAP1 gene, such that the mutationdecreases the expression of BAP1 mRNA, synthesis of BAP1 protein, orenzymatic activity of BAP1 when compared to the sequence of BAP1 from anon-tumor cell of the same individual. The presence of such a BAP1mutation indicates an increased risk for metastasis in the subject.Nucleotide and amino acid sequence mutations in tumor cells are detectedby comparison with the equivalent sequences from non-tumor cells fromthe same subject and/or by comparison to human wild type sequences SEQID NO: 3 (genomic nucleotide sequence) and SEQ ID NO: 1 (amino acidsequence). A mutation may also be identified by comparing cDNA sequencesobtained from mRNA in a tumor and non-tumor cell. “Wild type” cDNA mayhave the sequence SEQ ID NO: 2.

In another embodiment, the method comprises collecting a sample from asubject, and analyzing the level of BAP1 activity in the sample, where adecrease in BAP1 activity indicates an increased risk for metastasis inthe subject.

Each of these embodiments are discussed in more detail below.

(a) Analyzing the BAP1 Sequence to Determine Risk of Tumor Metastasis

One embodiment comprises analyzing the BAP1 nucleotide sequence and/orBAP1 amino acid sequence of a sample collected from a subject asdescribed in section (c) below. Typically, analyzing the BAP1 nucleotidesequence may comprise identifying a mutation in the BAP1 nucleotidesequence. As detailed above, “a mutation in the BAP1 sequence,” refersto a mutation in an exon of BAP1, an intron of BAP1, the promoter ofBAP1, the 5′ untranslated region of BAP1, the 3′ untranslated region ofBAP1, or any other regulatory region for the BAP1 gene (e.g. a spliceacceptor site), such that the mutation decreases the expression of BAP1mRNA, synthesis of BAP1 protein, or enzymatic activity of BAP1 whencompared to the sequence of BAP1 from a non-tumor cell of the sameindividual. Such a mutation may be a point mutation, a deletionmutation, or an insertion mutation. The mutation may be a missense ornonsense mutation. For instance, in one embodiment, the mutation maycause a premature truncation of BAP1. Alternatively, the mutation mayaffect a conserved amino acid in the ubiquitin carboxy-terminalhydrolase (UCH) domain or the UCH37-like domain (ULD) (for instance, seeFIG. 1B). Such a mutation may be identified using methods commonly knownin the art. For instance, see the Examples. Generally speaking, all or aportion of the BAP1 nucleic acid sequence may be sequenced and comparedto the wild-type genomic sequence (SEQ ID NO: 3) to identify a mutation.Alternatively or additionally, all or a portion of the BAP1 amino acidsequence may be compared to the wild-type amino acid sequence (SEQ IDNO: 1) to identify a mutation. Alternatively or additionally, all or aportion of cDNA obtained from BAP1 mRNA may be compared to the cDNAnucleotide sequence (RefSeq #NM_(—)004656; SEQ ID NO: 2).

However, with the knowledge of the mutations provided herein, it is aroutine matter to design detection means such as primers and/or probesthat would be able to detect and/or identify mutated sequences, such asmutated nucleotide sequences which differ from the wild-type SEQ ID NO:3 (or SEQ ID NO: 2, if cDNA is being examined), or mutated nucleotidesequences which differ from the BAP1 nucleotide sequence from anon-tumor cell of the subject. Possible techniques which might beutilized are well-established in the prior art and their use is readilyadaptable by the skilled person for the purposes of detecting the BAP1gene and/or BAP1 protein mutations disclosed herein. For example,amplification techniques may be used. Non-limiting examples ofamplification techniques may include polymerase chain reaction, ligasechain reaction, nucleic acid sequence based amplification (NASBA),strand displacement amplification (SDA), transcription mediatedamplification (TMA), Loop-Mediated Isothermal Amplification (LAMP),Q-beta replicase, Rolling circle amplification, 3SR, ramificationamplification (Zhang et al. (2001) Molecular Diagnosis 6 p141-150),multiplex ligation-dependent probe amplification (Schouten et al. (2002)Nucl. Ac. Res. 30 e57). Other related techniques for detecting mutationssuch as SNPs may include restriction fragment length polymorphism(RFLP), single strand conformation polymorphism (SSCP) and denaturinghigh performance liquid chromatography (DHPLC). A summary of many ofthese techniques can be found in “DNA Amplification: Currenttechnologies and applications” (Eds. Demidov & Broude (2004) Pub.Horizon Bioscience, ISBN:0-9545232-9-6) and other current textbooks.

A mutation of BAP1 may be an inactivating mutation, i.e., expressionlevels of BAP1 mRNA and/or synthesis of BAP1 protein are reduced and/orBAP1 protein activity is reduced in cells from a tumor sample from asubject, compared to expression level and/or synthesis level and/oractivity in cells from a non-tumor sample from the same subject. BAP1protein activity may be, for example, ubiquitin carboxy-terminalhydrolase activity.

In one embodiment, a mutation of BAP1 may be found in exon 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the BAP1nucleotide sequence. In another embodiment, a mutation of BAP1 may befound in the promoter of BAP1. In yet another embodiment, a mutation ofBAP1 may be found in the 5′ untranslated region. In still anotherembodiment, a mutation of BAP1 may be found in the 3′ untranslatedregion. In a certain embodiment, a mutation of BAP1 may be found in asplice acceptor site.

In particular embodiments, a mutation may be selected from one or moreof: deletion of the nucleotides equivalent to positions 3025-3074 of SEQID NO: 3; deletion of the nucleotides equivalent to positions 2026-2028of SEQ ID NO: 2; substitution of the nucleotide cytosine with thenucleotide guanine at the position equivalent to position 622 of SEQ IDNO: 2; substitution of the nucleotide guanine with the nucleotideadenine at the position equivalent to position 703 of SEQ ID NO: 2;substitution of the nucleotide cytosine with the nucleotide thymine atthe position equivalent to position 872 of SEQ ID NO: 2; deletion of thenucleotides equivalent to positions 960-968 of SEQ ID NO: 2; deletion ofthe nucleotides equivalent to positions 1083-1093 of SEQ ID NO: 2;substitution of the nucleotide adenine with the nucleotide guanine atthe position equivalent to position 2130 of SEQ ID NO: 2; deletion ofthe nucleotides equivalent to positions 3313-3335 of SEQ ID NO: 3;deletion of the nucleotides equivalent to positions 736-751 of SEQ IDNO: 2; insertion of the nucleotide adenine between positions equivalentto positions 1318 and 1319 of SEQ ID NO: 2; deletion of the nucleotidesequivalent to positions 468-487 of SEQ ID NO: 2 and insertion of thenucleotide adenine; deletion of nucleotide adenine at the positionequivalent to position 874 of SEQ ID NO: 2; deletion of the nucleotidesequivalent to positions 726-759 of SEQ ID NO: 3; substitution of thenucleotide thymine with the nucleotide adenine at the positionequivalent to position 2303 of SEQ ID NO: 2; deletion of the nucleotidesequivalent to positions 1829-1833 of SEQ ID NO: 2; deletion ofnucleotide cytosine at the position equivalent to position 259 of SEQ IDNO: 2; substitution of the nucleotide guanine with the nucleotidecytosine at the position equivalent to position 497 of SEQ ID NO: 2;substitution of the nucleotide cytosine with the nucleotide guanine atthe position equivalent to position 622 of SEQ ID NO: 2; deletion of thenucleotides equivalent to positions 2112-2120 of SEQ ID NO: 2;substitution of the nucleotide thymine with the nucleotide guanine atthe position equivalent to position 388 of SEQ ID NO: 2; deletion of thenucleotides equivalent to positions 2006-2017 of SEQ ID NO: 2; deletionof the nucleotides equivalent to positions 610-634 of SEQ ID NO: 2;deletion of the nucleotides equivalent to positions 739-776 of SEQ IDNO: 3; substitution of the nucleotide guanine with the nucleotidethymine at the position equivalent to position 7819 of SEQ ID NO: 3;substitution of the nucleotide cytosine with the nucleotide guanine atthe position equivalent to position 631 of SEQ ID NO: 2; deletion of thenucleotides equivalent to positions 2195-2220 of SEQ ID NO: 2;substitution of the nucleotide cytosine with the nucleotide thymine atthe position equivalent to position 221 of SEQ ID NO: 2. As outlinedabove, nucleotide numbering is by reference to the human wild-typesequences, for example, as represented by SEQ ID NO: 3 when comparinggenomic DNA or SEQ ID NO: 2 when comparing cDNA.

In a particular embodiment, a mutation may be a truncating mutation inexon 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 16 or 17 of BAP1, a missensemutation in exon 5, 6, 7 or 16, an in-frame deletion in exon 10, 15 or16, or a termination read-through in exon 17. In another particularembodiment, a BAP1 mutation may be a nonsense mutation in a BAP1 proteinencoded by the BAP1 nucleotide sequence, selected from Q36X, W196X, andQ253X. In yet another particular embodiment, a BAP1 mutation may be amissense mutation selected from C91W, G128R, H169Q, S172R or D672G. Instill another particular embodiment, an in-frame deletion may beselected from the group E283-S285del, E631-A634del or R666-H669del.Amino acid numbering is by reference to the human wild type sequences,for example, as represented by SEQ ID NO: 1.

(b) Analyzing the Level of BAP1 Activity

In other embodiments of the invention, the level of BAP1 activity in asample is analyzed. The “level of BAP1 activity” may refer to the levelof expression of BAP1 mRNA, the level of synthesis of BAP1 protein, orthe level of enzymatic activity of BAP1 in a sample.

In one embodiment, the level of BAP1 activity may refer to the level ofexpression of BAP1 mRNA in a sample. Generally speaking, if a sample hasa decreased level of expression of BAP1 mRNA, then the subject has anincreased risk of metastasis. In certain embodiments, the level of BAP1activity is decreased about 50% to about 100% compared to a non-tumorcell from the same individual. In other embodiments, the level of BAP1activity is decreased from about 60% to about 100% compared to anon-tumor cell from the same individual. In still other embodiments, thelevel of BAP1 activity is decreased from about 70% to about 95% comparedto a non-tumor cell from the same individual. In certain embodiments,the level of BAP1 activity is decreased about 100, 99, 98, 97, 96, 95,94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77,76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59,58, 57, 56, 55, 54, 53, 52, 51, or 50% compared to a non-tumor cell fromthe same individual.

Determining the level of expression of a BAP1 nucleic acid sequence,comprises, in part, measuring the level of BAP1 mRNA expression in atumor sample. Methods of measuring the level of mRNA in a tumor samplefor a particular nucleic acid sequence, or several sequences, are knownin the art. For instance, in one embodiment, the level of mRNAexpression may be determined using a nucleic acid microarray. Methods ofusing a nucleic acid microarray are well and widely known in the art. Inanother embodiment, the level of mRNA expression may be determined usingPCR. In these embodiments, the mRNA is typically reverse transcribedinto cDNA using methods known in the art. The cDNA may, for example,have nucleotide sequence SEQ ID NO: 2 when derived from mRNA obtainedfrom a non tumor cell. Methods of PCR are well and widely known in theart, and may include quantitative PCR, semi-quantitative PCR, multi-plexPCR, or any combination thereof. Other nucleic acid amplificationtechniques and methods are suggested above. In yet another embodiment,the level of mRNA expression may be determined using a TLDA (TaqMan lowdensity array) card manufactured by Applied Biosciences, or a similarassay. The level of mRNA expression may be measured by measuring anentire mRNA transcript for a nucleic acid sequence, or measuring aportion of the mRNA transcript for a nucleic acid sequence. Forinstance, if a nucleic acid array is utilized to measure the level ofmRNA expression, the array may comprise a probe for a portion of themRNA of the nucleic acid sequence of interest, or the array may comprisea probe for the full mRNA of the nucleic acid sequence of interest.Similarly, in a PCR reaction, the primers may be designed to amplify theentire cDNA sequence of the nucleic acid sequence of interest, or aportion of the cDNA sequence. One of skill in the art will recognizethat there is more than one set of primers that may be used to amplifyeither the entire cDNA or a portion of the cDNA for a nucleic acidsequence of interest. Methods of designing primers are known in the art.

Methods of extracting RNA from a tumor sample are known in the art. Forinstance, see Examples 1 and 2 of PCT/US09/041436, herein incorporatedby reference in its entirety.

The level of expression may or may not be normalized to the level of acontrol gene. Such a control gene should have a constant expression in atumor sample, regardless of the risk for metastasis of the tumor. Thisallows comparisons between assays that are performed on differentoccasions.

In another embodiment, the level of BAP1 activity may refer to the levelof BAP1 protein synthesis in a sample. Generally speaking, a decreasedlevel of BAP1 synthesis in a sample indicates an increased risk ofmetastasis in the subject. Methods of measuring the synthesis of BAP1are known in the art. For instance, immunofluorescence may be used, asdescribed in the Examples.

In yet another embodiment, the level of BAP1 activity may refer to thelevel of BAP1 enzymatic activity in a sample. Generally speaking, adecreased level of BAP1 enzymatic activity indicates an increased riskof metastasis in a subject. BAP1 has ubiquitin carboxy-terminalhydrolase activity. Such activity may be measured using methods wellknown in the art. See, for instance, Scheuermann J C, et al: Histone H2Adeubiquitinase activity of the Polycomb repressive complex PR-DUB,Nature 2010, 465:243-247 (the measurement of histone H2Amonoubiquitination); Machida Y J, et al: The deubiquitinating enzymeBAP1 regulates cell growth via interaction with HCF-1, J Biol Chem 2009,284:34179-34188 (the measurement of HCFC1 deubiquitination); Russell NS, Wilkinson K D. Deubiquitinating enzyme purification, assayinhibitors, and characterization. Methods Mol Biol 2005; 301:207-19(other strategies for measurement of deubiquitinating enzymatic activityusing substrates that can be monitored, such as described in Russell etal.).

(c) Collecting a Sample from a Subject

A method of the invention comprises, in part, collecting a sample from asubject. Suitable samples comprise one or more tumor cells, either froma primary tumor or a metastasis. In one embodiment, a suitable samplecomprises a melanoma cell. In another embodiment, a suitable samplecomprises a carcinoma cell. In yet another embodiment, a suitable samplecomprises a sarcoma cell. In an exemplary embodiment, a suitable samplecomprises a uveal melanoma cell. In another exemplary embodiment, asuitable sample comprises a cutaneous melanoma cell. In someembodiments, a suitable sample may be a circulating tumor cell.Circulating tumor cells may be found in a bodily fluid (e.g. plasma,sputum, urine, etc.) or other excrement (e.g. feces).

Methods of collecting tumor samples are well known in the art. Forinstance, a tumor sample may be obtained from a surgically resectedtumor. In uveal melanoma, for example, a tumor sample may be obtainedfrom an enucleation procedure. Alternatively, the tumor sample may beobtained from a biopsy. This is advantageous when the tumor is smallenough to not require resection. In an exemplary embodiment, the tumorsample may be obtained from a fine needle biopsy, also known as a needleaspiration biopsy (NAB), a fine needle aspiration cytology (FNAC), afine needle aspiration biopsy (FNAB) or a fine needle aspiration (FNA).A tumor sample may be fresh or otherwise stored so as to reduce nucleicacid degradation. For instance, a tumor sample may be a fresh frozentumor sample or a formalin-fixed paraffin embedded tumor sample.

In certain embodiments, the method of the invention may be performedwith a tumor sample comprising about five cells or less. In oneembodiment, the tumor sample may comprise about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more cells. In another embodiment, thetumor sample may comprise 20, 25, 30, 35, 40 or more cells.

(d) Determining the Risk of Metastasis

A method of the invention further comprises determining the risk ofmetastasis. The level of risk is a measure of the probability of ametastasis occurring in a given individual. If a mutation is identified,as described in section (a) above, in a sample from a subject, then thesubject is at a higher risk (i.e., there is an increased probability) ofdeveloping metastases then a subject without a mutation in a BAP1nucleotide sequence and/or BAP1 amino acid sequence. Alternatively, ifthe level of BAP1 activity is decreased, as described in section (b)above, then the subject is at a higher risk of developing metastasesthen a subject with out a decreased level of BAP1 activity. Forinstance, the risk may be greater than about 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In someembodiments, the risk may be greater than about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%. In particular embodiments, the risk maycontinue to increase over time. For example, the risk may be about 50%at five years after initial cancer diagnosis and 90% for ten years.

Alternatively, if a mutation in not identified (i.e. the BAP1 nucleotideand corresponding amino acid sequence is wild-type) in a sample from asubject, then the subject is at lower risk of developing metastases.Similarly, if the level of BAP1 activity is not decreased, then thesubject is at a lower risk of developing metastasis. For instance, therisk may be less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments,the risk may be less than about 20%, 15%, 10%, or 5%. In particularembodiments, the risk may be low, but may still increase over time. Forexample, the risk may be about 5% at five years and 10% at ten years.

Increased or decreased “risk” or “probability” may be determined, forexample, by comparison to the average risk or probability of anindividual cancer patient within a defined population developingmetastasis. For example, by way of illustration, for a given cancer theoverall proportion of patients who are diagnosed with a metastasiswithin 5 years of initial cancer diagnosis may be 50%. In thistheoretical context, an increased risk for an individual will mean thatthey are more than 50% likely to develop a metastasis within 5 years,whereas a reduced risk will mean that they are less than 50% likely todevelop a metastasis. Such comparisons may, in some circumstances, bemade within patient populations limited or grouped using other factorssuch as age, ethnicity, and/or the presence or absence of other riskfactors.

(e) Combination of Methods

In certain embodiments, a method of the invention may be used inconjunction with a method as described in PCT/US09/041436, hereinincorporated by reference in its entirety, to determine the risk ofmetastasis in a subject.

II. Method for Detecting a Metastasis

Another aspect of the present invention is a method for detecting thepresence of a metastasis. In one embodiment, the method generallycomprises collecting a sample from a subject, analyzing the BAP1nucleotide and/or BAP1 amino acid sequence in the sample, anddetermining the presence of a mutation in the BAP1 sequence. Thepresence of the mutation indicates the presence of a metastasis. Asoutlined above, the presence of a mutation may be determined bycomparison of a sequence from a tumor cell with a sequence from anon-tumor cell from the same subject and/or by comparison to SEQ ID NO:1 (wild type amino acid sequence) or SEQ ID NO: 3 (wild type genomic DNAsequence). It may also be determined by obtaining cDNA from BAP1 mRNA inthe cell and comparing the sequence to SEQ ID NO: 2.

In another embodiment, the method comprises collecting a sample from asubject, and analyzing the level of BAP1 activity in the sample, where adecrease in BAP1 activity indicates the presence of a metastasis in thesubject. Suitable samples, methods of analyzing a BAP1 nucleotidesequence and/or BAP1 amino acid sequence, and methods of determining thelevel of BAP1 activity in a sample are described in section I above.

III. Biomarker for Metastasis

Yet another aspect of the invention encompasses a biomarker for tumormetastasis. In one embodiment, a biomarker of the invention comprises amutation in a BAP1 nucleotide sequence and/or BAP1 amino acid sequence,as described in section I(a) above. In another embodiment, a biomarkerof the invention comprises a decreased level of BAP1 activity, asdescribed in section I(b) above. This may include a decrease in BAP1protein synthesis. Where the biomarker is a BAP1 amino acid sequencecomprising a mutation, the presence of the biomarker may be detected byuse of an antibody which specifically binds to the biomarker. Suchantibodies are encompassed within the scope of the present invention, aswell as kits comprising the antibody and methods of use thereof. In eachof the above embodiments, a tumor may be a melanoma, carcinoma, orsarcoma. In an exemplary embodiment, the tumor is a melanoma. In afurther exemplary embodiment, the tumor is a uveal melanoma. In yetanother exemplary embodiment, the tumor is a cutaneous melanoma.

DEFINITIONS

As used herein, “carcinoma” refers to a malignant tumor derived from anepithelial cell. Non-limiting examples of carcinoma may includeepithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma,basal cell neoplasms, basal cell carcinoma, transitional cellcarcinomas, adnexal and skin appendage neoplasms, mucoepidermoidneoplasms, cystic, mucinous and serous neoplasms, ductal, lobular andmedullary neoplasms, acinar cell neoplasms, complex epithelialneoplasms, squamous cell carcinoma, adenosquamous carcinoma, anaplasticcarcinoma, large cell carcinoma, small cell carcinoma, andadenocarcinomas such as adenocarcinoma, linitis plastica, vipoma,cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma,and grawitz tumor.

As used herein, “melanoma” refers to a malignant tumor of a melanocyte.In one embodiment, the melanoma may be a uveal melanoma. In anotherembodiment, the melanoma may be a cutaneous melanoma. In anotherembodiment, the melanoma may be a mucosal melanoma.

As used herein, “regulatory region” refers to a nucleic acid sequenceoperably linked to a nucleic acid encoding BAP1 such that the regulatoryregion modulates the transcription of BAP1 mRNA.

As used herein, “sarcoma” refers to a malignant tumor derived fromconnective tissue. Non limiting examples of a sarcoma may includeAskin's Tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma,primitive neuroectodermal tumor (PNET), malignant hemangioendothelioma,malignant peripheral nerve sheath tumor (malignant schwannoma),osteosarcoma and soft tissue sarcomas such as alveolar soft partsarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma,desmoid Tumor, desmoplastic small round cell tumor, epithelioid sarcoma,extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma. Lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.

As used herein, “subject” refers to a mammal capable of being afflictedwith a carcinoma, melanoma, or sarcoma, and that expresses a homolog toBAP1. In addition to having a substantially similar biological function,a homolog of BAP1 will also typically share substantial sequencesimilarity with the nucleic acid sequence of BAP1. For example, suitablehomologs preferably share at least 30% sequence homology, morepreferably, 50%, and even more preferably, are greater than about 75%homologous in sequence. In determining whether a sequence is homologousto BAP1, sequence similarity may be determined by conventionalalgorithms, which typically allow introduction of a small number of gapsin order to achieve the best fit. In particular, “percent homology” oftwo polypeptides or two nucleic acid sequences may be determined usingthe algorithm of Karlin and Altschul [(Proc. Natl. Acad. Sci. USA 87,2264 (1993)]. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (J. Mol. Biol. 215, 403 (1990)).BLAST nucleotide searches may be performed with the NBLAST program toobtain nucleotide sequences homologous to a nucleic acid molecule of theinvention. Equally, BLAST protein searches may be performed with theXBLAST program to obtain amino acid sequences that are homologous to apolypeptide of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul, et al.(Nucleic Acids Res. 25, 3389 (1997)). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are employed. See www.ncbi.nlm.nih.gov for moredetails. In an exemplary embodiment, the subject is human. In certainembodiments, the subject may have a carcinoma, sarcoma, or melanoma. Inother embodiments, the subject may be suspected of having a carcinoma,sarcoma, or melanoma.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. Those of skill in the art should, however, in light ofthe present disclosure, appreciate that may changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

Examples

The following examples illustrate various iterations of the invention.

Example 1 BAP1 Mutations and Uveal Melanoma Metastasis

Uveal melanoma (UM) is the most common primary cancer of the eye and hasa strong propensity for fatal metastasis (1). UMs are divided into class1 (low metastatic risk) and class 2 (high metastatic risk) based on avalidated multi-gene clinical prognostic assay included in the TNMclassification system (2, 3). However, the genetic basis of metastasisremains unclear. Oncogenic mutations in the Gα_(q) stimulatory subunitGNAQ are common in UM (4), but these mutations occur early intumorigenesis and are not correlated with molecular class or metastasis(5, 6). On the other hand, class 2 tumors are strongly associated withmonosomy 3 (7), suggesting that loss of one copy of chromosome 3 mayunmask a mutant gene on the remaining copy which promotes metastasis.

Using exome capture followed by massively parallel sequencing (8, 9), weanalyzed two class 2 tumors that were monosomic for chromosome 3 (MM56and MM70) and matching normal DNA from peripheral blood lymphocytes.Both tumors contained inactivating mutations in BAP1, located atchromosome 3p21.1 (FIG. 1A). MM56 contained a C/G to T/A transition thatcreated a premature termination codon (p.W196X). MM70 contained adeletion of 11 bp in exon 11, leading to a frameshift and prematuretermination of the BAP1 protein (p.Q322fsX100). The matched normal DNAsamples did not contain these mutations, indicating that they werelikely to be somatic in origin. No gene on chromosome 3 other than BAP1contained deleterious somatic mutations that were present in both tumors(Table 1).

TABLE 1 Summary of DNA sequence alterations identified by exomecapture and massively parallel sequencing of tumor DNA andatching normal peripheral blood lymphocyte DNA from uvealmelanomas MM056 and MM070 Chr 3 Indel Ref- Ref. location End erenceAmino New (hg19) Tumor (hg19) Gene Codon Acid Codon 20,216,514 MM56SGOL1 GGA G GTA 43,641,970 MM70 AN010 CTA L CTG 44,488,382 MM56 ZNF445AGC S AGA 44,636,130 MM70 ZNF660 CTT L ATT 45,715,863 MM56 LIMD1 TCC STAC 46,008,495 MM70 FYCO1 CAG Q CAT 48,457,135 MM56 PLXNB1 TGT C TGC48,630,022 MM56 COL7A1 GTG V GTT 49,690,418 MM56 BSN CCT P CCA49,699,662 MM56 BSN TGG W GGG 52,439,264  MM70* 52,439,274 BAP1 deletion52,439,264  MM70* BAP1 CAC H CAG 52,439,266  MM70* BAP1 CAC H AAC52,440,916 MM56 BAP1 TGG W TGA 52,814,305 MM56 ITIH1 GAG E GAT56,650,054 MM70 CCDC66 TCT S CCT 123,695,755 MM70 ROPN1 CGG R TGG135,825,122 MM70 PPP2R3A ACG T ATG 172,351,305 MM56 NCEH1 ACT T AAT180,327,975 MM70 TTC14 GGA G GAA 183,041,104 MM56 MCF2L2 GGC G GGA194,062,926 MM56 CPN2 ACC T AAC 194,408,437 MM56 FAM43A GAG E GAT195,306,227 MM70 APOD AAT N GAT Present on Chr 3 New Ref Sanger locationAmino base Con- Read sequence (hg19) Acid (hg19) sensus Depthvalidation2 20,216,514 V C M 16 No 43,641,970 L T Y 77 Not done44,488,382 R G K 14 Not done 44,636,130 I C M  9 No 45,715,863 Y C M 53No 46,008,495 H C M 19 Not done 48,457,135 C A R 11 Not done 48,630,022V C M  9 Not done 49,690,418 P T W 11 No 49,699,662 G T K 21 No52,439,264 CCCC deleted 19 Yes ATCC CAC (SEQ ID NO: 5) 52,439,264 Q G Q15 Yes 52,439,266 N G N 13 Yes 52,440,916 X C X 27 Yes 52,814,305 D G K15 No 56,650,054 P T Y 13 No 123,695,755 W G R 39 Yes (germline)135,825,122 M C Y 33 Yes (somatic) 172,351,305 N G K 20 No 180,327,975 EG K 22 No 183,041,104 G G K 25 Not done 194,062,926 N G K 24 No194,408,437 D G K  9 No 195,306,227 D T Y 21 Not done ¹In the case ofInsertion/Deletions (InDels) that were detected with Novoalign, column 1defnes the start and column 3 defines the end. The BAP1 mutationsreported in the current manuscript are asterisked and were incorrectlydetected as base substitutions in the case of the InDel in MM70, inaddition to being correctly detected as an InDel with Novoalign. This iswhy several substitutions are reported in MM70 for this gene, althoughthey correspond to a single mutational event. We detected 20 additionalputative somatic mutations in genes on chromosome 3. The predicted codonand amino acid changes for the appropriate strand are indicated whereapplicable, along with the base in the hg19 reference sequence and thebase change reported as a consensus using IUPAC nomenclature. Referencebases and reference base changes are reported for the plus strand. Depthrefers to the read depth of the altered base in the tumor sample. Sangerresequencing was performed to validate each variant detected in thetumor but not the germline. 2In the case of one mutation residing inROPN1 the mutation was confirmed in the tumor and was also seen in theblood. This had been missed with exome capture. In the case of onemutation residing in PPP2R3A in tumor MM070 the mutation was confirmedto be a somatic alteration.

BAP1 encodes a nuclear ubiquitin carboxy-terminal hydrolase (UCH), oneof several classes of deubiquitinating enzymes (10). In addition to theUCH catalytic domain, BAP1 contains a UCH37-like domain (ULD) (11),binding domains for BRCA1 and BARD1, which form a tumor suppressorheterodimeric complex (12), and a binding domain for HCFC1, whichinteracts with histone-modifying complexes during cell division (11, 13,14). BAP1 also interacts with ASXL1 to form the Polycomb grouprepressive deubiquitinase complex (PR-DUB), which is involved in stemcell pluripotency and other developmental processes (15, 16). BAP1exhibits tumor suppressor activity in cancer cells (10, 12), and BAP1mutations have been reported in a small number of breast and lung cancersamples (10, 17).

To further investigate BAP1, genomic DNA from 29 additional class 2 UMs,and 26 class 1 UMs were subjected to Sanger re-sequencing of all BAP1exons. Altogether, BAP1 mutations were identified in 26 of 31 (84%)class 2 tumors, including 13 out-of-frame deletions and two nonsensemutation leading to premature protein termination, six missensemutations, four in-frame deletions, and one mutation predicted toproduce an abnormally extended BAP1 polypeptide (FIG. 1A-C). Three ofthe missense mutations affected catalytic residues of the UCH activesite (C91 and H169), two occurred elsewhere in the UCH domain, and oneaffected the ULD (FIG. 1B-C). All BAP1 missense mutations and in-framedeletions affected phylogenetically conserved amino acids (FIG. 10).Only one of 26 class 1 tumors contained a BAP1 mutation (NB101). Thiscase may represent a transition state in which the tumor has sustained aBAP1 mutation but has not yet converted to class 2, suggesting that BAP1mutations may precede the emergence of the class 2 signature. SomaticBAP1 mutations were also detected in two of three metastatic tumors. Thesummary of genetic data on uveal melanoma tumor samples are presented inTables 2 and 3.

TABLE 2 Summary genetic data on uveal melanoma tumor samples in thestudy Source BAP1 of Gene Loss mutation BAP1 Tumor tumor expression ofIn normal Mutation Number analyzed class Chr 3 DNA In Tumor MM 010Primary Class 1 No No No MM 016 Primary Class 1 No No No MM 018 PrimaryClass 1 No No No MM 050 Primary Class 1 No No No MM 074 Primary Class 1No No No MM 086 Primary Class 1 No No No MM 089 Primary Class 1 No No NoMM 092 Primary Class 1 No No No MM 101 Primary Class 1 No No No MM 109Primary Class 1 No No No MM 113 Primary Class 1 No No No MM 122 PrimaryClass 1 Yes No No NB 092 Primary Class 1 Yes No No NB 096 PrimaryClass 1 No No No NB 099 Primary Class 1 No No No NB 101 Primary Class 1No No Yes NB 102 Primary Class 1 No No No NB 104 Primary Class 1 Yes NoNo NB 107 Primary Class 1 No No No NB 108 Primary Class 1 No No NoNB 109 Primary Class 1 No No No NB 112 Primary Class 1 No No No NB 113Primary Class 1 No No No NB 116 Primary Class 1 Yes No No NB 119 PrimaryClass 1 No No No NB 126 Primary Class 1 No No No MM 046 Primary Class 2Yes No Yes MM 054 Primary Class 2 Yes No Yes MM 055 Primary Class 2 YesNo Yes MM 056 Primary Class 2 Yes No Yes MM 060 Primary Class 2 Yes NoYes MM 066 Primary Class 2 Yes No Yes MM 070 Primary Class 2 Yes No YesMM 071 Primary Class 2 Yes No Yes MM 080 Primary Class 2 Yes No NoMM 081 Primary Class 2 Yes No Yes MM 083 Primary Class 2 Yes No YesMM 087 Primary Class 2 Yes Yes Yes MM 090 Primary Class 2 Yes No YesMM 091 Primary Class 2 Yes No Yes MM 100 Primary Class 2 Yes No YesMM 103 Primary Class 2 Yes No Yes MM 110 Primary Class 2 Yes NO YesMM 120 Primary Class 2 Yes No Yes MM 121 Primary Class 2 Yes No YesMM 125 Primary Class 2 Yes No Yes MM 127 Primary Class 2 No No No MM 128Primary Class 2 Yes No Yes MM 133 Primary Class 2 No No No MM 134Primary Class 2 No No No MM 135 Primary Class 2 Yes No Yes NB 185Primary Class 2 No No Yes NB 191 Primary Class 2 No No Yes NB 195Primary Class 2 No No Yes NB 199 Primary Class 2 No No Yes NB 200Primary Class 2 No No Yes NB 214 Primary Class 2 No No No MM 152MMetastasis NO NO No Yes NB 07M Metastasis Class 2 Yes No Yes PV L8Metastasis No No No No BAP1 Mutation in Exon Predicted Tumor cDNA ofWith Protein effect on Number gDNA (hg19) mutation change protein MM 010MM 016 MM 018 MM 050 MM 074 MM 086 MM 089 MM 092 MM 101 MM 109 MM 113MM 122 NB 092 NB 096 NB 099 NB 101 g chr3.52,441,485-  6 UnknownLoss of  52,441,436delTCCCCGT splice AGAGCAAAGGATATGC acceptor ofGATTGGCAATGCCCCG exon 6 and GAGTTGGCAA potential (SEQ ID NO: 4) cryptic splice leading to out of frame peptide and premature termination NB 102NB 104 NB 107 NB 108 NB 109 NB 112 NB 113 NB 116 NB 119 NB 126 MM 046C 2026-2028delGTG 15 P Deletion of K637_C63 K637 and 8delinsN C638 andsubstitution of N MM 054 G chr3 52,441,434-  6 Unknown Los of 52,441,483del splice acceptor of exon 6 and potential cryptic splice leading to out of frame peptide and premature termination MM 055c 622C > G  7 pH169Q UCH active site mutated MM 056 c 703G > A  8 pW196XPremature Termination MM 060 c 872C > T  9 pQ253X Premature TerminationMM 066 c 960- 10 P. E283- In-frame 968delCTGAGGAGT S285del deletionbetween  BARD1 and HCFC1 binding domains MM 070 c 1083- 11 P Premature1093delCCCCatCCCAC Q322fsx100 Termination (SEQ ID NO: 5) MM 071c 2130A > G 16 Pd72G AA change in ULD domain MM 080 MM 081g chr3.52441197- 7 unknown Loss of 52441174delTGACCATG spliceGTAGGCACCATGAGC acceptor of (SEQ ID NO: 6) exon 7 and potential cryptic splice  leading to  out of frame peptide and premature terminationMM 083 c 736- 8 pR207fsX32 Premature 751delCGGGTCATCATG termination GAG(SEQ ID NO: 7) MM 087 c 1318-1319insA 12 pE402fsX2 Premature terminationMM 090 c 468-487delinsA 5 p.F118X Premature termination MM 091 c 874delG9 pQ253fs Premature termination MM 100 g chr3 52,443,784- 2 UnknownLoss of 42,443,750del splice CCCCTCCTCTTGTCGC acceptor ofCCCACCCAGGCCTCTT exon 2 CAC and potential (SEQ ID NO: 8) cryptic splice  leading to  out of frame peptide and premature terminationMM 103 c 2303T > A 17 pTer729R Read through termination codon MM 110c 1829-1833delCCCCT 13 ps571fsX25 Premature termination MM 120 C 259delC4 pF48fsX22 Premature termination MM 121 c 497G > C 6 pG128R MissenseMM 125 c 622C > G 7 pH169Q* UCH active site mutated MM 127 MM128c 2112-2120del9 16 R666- RRTH GAAGGACCC H669 deletion in delinsNULD domain MM 133 MM 134 MM 135 c 388T > G 5 p.C91W UCH activesite mutated (active site) NB 185 c 2006-2017 15 p E631- InternaldelGAGCTGCTGGCA A634del in-frame (SEQ ID NO: 9) deletion in ULD domainNB 191 C 610-634 7 P Premature delGGAGGCGTTCCACT M166fsX12 terminationTTGTCAGCTAT (SEQ ID NO: 10) NB 195 g chr3 52,443,771- 2 Unknown Loss of52,443,734 splice delCGCCCCACCCAGGC acceptor of CTCTTCACCCTGCTCG exon 2TGGAAGAT and potential (SEQ ID NO: 11) cryptic  splice  leading to out of frame peptide and premature termination NB 199 chr3:52,436,691G >T 16 Unknown Unknown, Splice acceptor AG  likely to AT prematuretermination NB 200 c 631C > G 7 p S172R Missense NB 214 MM 152MC 2195-2220 17 p Premature delCAGAACCATCTCCG E693fsX13 terminationTGCGGCGGCGCCA (SEQ ID NO: 12) NB 07M c 221C > T 3 Q36* Prematuretermination PV L8

TABLE 3 BAP1 muta- BAP1 Source of tion muta- Mutation Tumor tumor GEPnormal tion in gDNA Number analyzed class LOH3 DNA tumor (hg19) MM 133Primary/fresh 2 ? NA No NA frozen MM 134 Primary/fresh 2 ? NA No NAfrozen MM 137 Primary/fresh 2 ? No Yes g.chr3:52443889- frozen52443927delATTC ATCTTCCCGCGG GGCGGCCCCTC AGCGCCATGTCC (SEQ ID NO: 13)MM 138 Primary/fresh 2 ? NA No NA frozen MM 144 Primary/fresh 2 ? No Yesc.265delC; frozen g.chr3:52442595 delC MM 150 Primary/fresh 2 ? NA No NAfrozen MM 151A Primary/fresh 2 ? No Yes g.chr3:52440925- frozen52440918delAGG GCCCT Mouse ? NA Yes g.chr3:52440925- 204 52440918delAGG(MM151 GCCCT A met) MM 161 Primary/fresh 2 ? ? Yes c.1013-1014delAG;frozen g.chr3:52439814- 52439813delAG MM 162 Primary/fresh 2 ? ? Yesg.chr3:52437431 frozen G > C and chr3:52437433 delA  OP-11-Primary/paraf ? ? Yes g.chr3: 52442086- 953 fin embedded 52442106delGGTA(Emory) TCAGCTGTGAAA CCAAG (SEQ ID NO: 14) MM 131T Primary/fresh  1b ?NA No NA frozen MM 159T Primary/fresh 2 ? NA No NA frozen PredictedTumor BAP1 Mutant Protein Predicted effect Number cDNA exon changeon protein MM 133 NA NA MM 134 NA NA MM 137 c.82- 1 prematuredeletes first two 121del truncation  aa (MN) and 33 bp from 5′UTR(ATTCATCTTCCCGCG GGGCGGCCCCTCAG CGCCATGTCC) (SEQ ID NO: 13) MM 138 NA NAMM 144 c.265delC 4 premature truncation (p.F50LfsX 22) MM 150 NA NAMM 151A 8 Deletion of delete AG splice  exon 6 donor of exon 8 and then deletion  of 6 bp in exon 6- leaves 48 bp. Mightbe exon skipping. Mouse 8 Deletion  204 of exon 6 (MM151 A met) MM 16110 premature Premature  truncation termination MM 162 13 prematureSplice mutation, truncation  deletion of A OP-11- 10 premature 953truncation (Emory) MM 131T MM 159T NA NA NA: Not applicable

One copy of chromosome 3 was missing in all 17 BAP1-mutant class 2tumors for which cytogenetic data were available, consistent withchromosome 3 loss uncovering recessive BAP1 mutations. Normal DNA from20 patients with BAP1-mutant class 2 primary tumors and the two withmetastatic tumors was available and did not contain a BAP1 mutation,indicating that the mutations were somatic in origin. However, wedetected one germline mutation (p.E402fsX2; c.1318-1319insA) in thepatient with the class 1 tumor NB101 (Table 2), and this case wasparticularly interesting. Re-sequencing of this tumor revealed adeletion of a segment of exon 6 of BAP1, including its splice acceptor.This mutation is predicted to result in a premature truncation of theencoded protein (Table 2). However, the wildtype allele was present atlevels similar to the mutant allele, indicating that it was disomic forchromosome 3 (FIG. 2). Hence, this case may represent a transition statein which the tumor is still class 1 but has sustained a BAP1 mutation.This might suggest that the BAP1 mutations precede loss of chromosome 3and the emergence of the class 2 signature during tumor progression.Thus, germline alterations in BAP1 can predispose to UM.

Other germline (blood) mutations in exon 13 (g.chr3:52437465insT;pE566X; c.1695-1696insT leading to premature protein termination) inFUM1-01 and FUM-02 were also detected (see FIG. 18).

GNAQ mutation status was available in 15 cases. GNAQ mutations werepresent in 4/9 BAP1 mutant tumors and 3/6 BAP1 wildtype tumors,indicating that there was no correlation between GNAQ and BAP1 mutationstatus.

UM usually metastasizes to the liver, where it is difficult to obtainspecimens for research. However, we were able to obtain sufficient DNAfrom three UM liver metastases for analysis. BAP1 mutations weredetected in two of the three metastatic tumors, supporting thehypothesis that cells mutant for BAP1 are indeed the ones responsiblefor metastasis (Table 2). NB071M contained a nonsense mutation (Q36X),and MM152M contained an out-of-frame deletion (p.E693fsX13). Bothmutations are predicted to cause premature protein truncation. Primarytumor DNA on either case was unavailable.

Quantitative RT-PCR showed that BAP1 mRNA levels were significantlylower in class 2 tumors compared to class 1 tumors (P<0.0001) (FIG. 3A).Truncating mutations were associated with significantly lower mRNAlevels than missense mutations (P=0.001) (FIG. 3B), consistent withnonsense mediated mRNA decay in the former group. Class 2 tumors inwhich BAP1 mutations were not identified expressed very low levels ofBAP1 mRNA (FIG. 3B).

To determine whether the low BAP1 mRNA levels in class 2 tumors withoutdetectable BAP1 mutations may be explained by DNA methylation, weperformed a preliminary analysis of DNA methylation of BAP1. This didnot reveal a convincing difference between class 1 and class 2 tumors.However, analysis of the BAP1 promoter was limited by an unusuallycomplex CpG island that will require further work to resolve. Thus, wecannot rule out a role for methylation in class 2 tumors in which BAP1mutations were not found. However, with almost 85% of class 2 tumorsharboring mutations, we do not expect that methylation will be a majormechanism of BAP1 inactivation. An alternative explanation is that thesetumors may contain very large deletions of the BAP1 locus or othermutations not detectable by our sequencing method.

Immunofluorescence revealed abundant nuclear BAP1 protein in two class 1tumors but virtually none in four BAP1 mutant class 2 tumors (FIG. 4).This was expected for the two tumors with mutations expected to causepremature protein terminations (MM 091 and MM 100), but it wassurprising for the two tumors with missense mutations (MM 071 and MM135) and suggests that these mutations lead to protein instability.

RNAi-mediated knock down of BAP1 in 92.1 UM cells, which did not harbora detectable BAP1 mutation, recapitulated many characteristics of thede-differentiated class 2 UM phenotype (18). Cells transfected withcontrol siRNA exhibited typical melanocytic morphology, includingdendritic projections and cytoplasmic melanosomes (FIG. 5), whereascells transfected with BAP1 siRNA lost these features, developed arounded epithelioid morphology and grew as multicellular non-adherentspheroids, strikingly similar to the features of class 2 clinical biopsysamples (FIG. 5). Microarray gene expression profiling of 92.1 UM cellstransfected with control versus BAP1 siRNA showed that most of the topgenes that discriminate between class 1 and class 2 tumors shifted inthe class 2 direction in BAP1 depleted cells compared to control cells(FIG. 6). Similarly, depletion of BAP1 shifted the gene expressionprofile of the multi-gene clinical prognostic assay towards the class 2signature (FIG. 7A). BAP1 depletion caused a reduction in mRNA levels ofneural crest migration genes (ROBO1), melanocyte differentiation genes(CTNNB1, EDNRB and SOX10) and other genes that are down-regulated inclass 2 tumors (LMCD1 and LTA4H) (18). In contrast, BAP1 depletioncaused an increase in mRNA levels of CDH1 and the proto-oncogene KIT,which are highly expressed in class 2 tumors (19). Similarly, mRNAtranscripts of KIT, MITF and PAX3, whose protein products are associatedwith proliferation of pre-terminally differentiated melanocytes and haveoncogenic effects when overactive in melanoma (20-22), weresignificantly up-regulated by BAP1 depletion (FIG. 7B). Similar resultswere seen in other UM cell lines and with an independent BAP1 siRNA(FIG. 7C).

GNAQ mutations occur early in UM and are not sufficient for malignanttransformation (4), but they may create a dependency of the tumor cellson constitutive GNAQ activity. In contrast, BAP1 mutations occur laterin UM progression and coincide with the onset of metastatic behavior.Thus, simultaneous targeting of both genetic alterations might havesynergistic therapeutic effects. One potential strategy to counteractthe effects of BAP1 mutation would be to inhibit the RING1 ubiquinatingactivity that normally opposes the deubiquinating activity BAP1 (16).Our findings strongly implicate mutational inactivation of BAP1 as a keyevent in the acquisition of metastatic competence in UM, and theydramatically expand the role of BAP1 and other deubiquitinating enzymesas potential therapeutic targets in cancer.

Materials and Methods for Example 1. Patient Materials:

Acquisition of patient material (matched tumor and normal samples) hasbeen described elsewhere (25) (Table 4). This study was approved by theHuman Studies Committee at Washington University (St. Louis, Mo.), andinformed consent was obtained from each subject. Tumor tissue wasobtained immediately after eye removal, snap frozen, and prepared forRNA and DNA analysis. UM metastases were collected from liver biopsiesat the time of metastatic diagnosis. All samples werehistopathologically verified. Genomic DNA from tumors was prepared usingthe Wizard Genomic DNA Purification kit (Promega, Madison, Wis.). DNAfrom blood was isolated using the Quick Gene DNA whole blood kit S(Fugifilm, Tokyo, Japan). RNA was isolated using the PicoPure kit(including the optional DNase step). All RNA samples were converted tocDNA using the High Capacity cDNA Reverse Transcription kit from AppliedBiosystems (Applied Biosystems Inc., Foster City, Calif.) following themanufacturer's protocol.

TABLE 4 Summary of clinical and pathologic data on uveal melanomapatients in the study Age at Tumor Source primary Tumor thicknessPathologic of tumor diameter of Ciliary cell type of Mons Tumor tumordiag- of primary primary body primary Treatment of follow- Numberanalyzed nosis Gender tumor tumor involvement tumor primary tumor upMetastasis MM 010 Primary 41 Male 17 9.9 No Mixed Enucleation 131.2 YesMM 016 Primary 24 Female 24 12.6 Yes Spindle Enucleation 87.8 Yes MM 018Primary 55 Male 12 9.2 No Epithelioid Enucleation 67.4 No MM 050 Primary50 Female 19 8.9 Yes Epithelioid Enucleation 71.4 No MM 074 Primary 77Male N/A 22.0 N/A Mixed Enucleation 24.4 No MM 086 Primary 47 Male 1414.0 No Spindle Enucleation 17.7 No MM 089 Primary 74 Male 18 8.1 YesSpindle Enucleation 8.1 Yes MM 092 Primary 61 Male 20 13.4 YesEpithelioid Enucleation 8.0 No MM 101 Primary 66 Female 15 6.4 NoSpindle Enucleation 10.8 No MM 109 Primary 56 Male 15 12.7 Yes SpindleEnucleation 8.8 No MM 113 Primary 54 Male 14 11.0 No Mixed Enucleation8.0 No MM 122 Primary 52 Male N/A N/A Yes Epithelioid Enucleation 1.0 NoNB 092 Primary 53 Male 11 4.4 No Mixed Brachytherapy 25.2 No NB 096Primary 55 Female 15 3.8 No Spindle Brachylerapy 24.1 No NB 099 Primary76 Male N/A N/A No Other Biopsy 1.0 No NB 101 Primary 57 Female 14 2.6Yes Other Brachytherapy 23.2 No NB 102 Primary 83 Female 13 2.4 NoEpithelioid Brachytherapy 27.5 No NB 104 Primary 62 Male 13 5.5 NoEpithelioid Brachytherapy 24.1 No NB 107 Primary 58 Male 15 10.0 YesEpithelioid Brachytherapy 19.9 No NB 108 Primary 66 Female 10 2.6 NoOther Brachytherapy 17.8 No NB 109 Primary 76 Male 18 8.4 Yes SpindleBrachytherapy 9.1 No NB 112 Primary 53 Male 12 6.1 No OtherBrachytherapy 21.4 No NB 113 Primary 70 Male 14 3.2 Yes SpindleBrachytherapy 13.2 No NB 116 Primary 85 Female 17 7.1 Yes SpindleBrachytherapy 13.4 No NB 119 Primary 69 Female 16 5.9 Yes SpindleBrachytherapy 21.8 No NB 126 Primary 34 Female 17 8.1 No SpindleBrachyterhapy 22.4 No MM 046 Primary 69 Female 22 9.0 Yes EpithelioidEnucleation 32.6 Yes MM 054 Primary 80 Female 15 6.7 No MixedEnucleation 34.6 Yes MM 055 Primary 82 Female 19 8.6 Yes EpithelioidEnucleation 81.3 Yes MM 056 Primary 63 Male 18 11.7 Ye EpithelioidEnucleation 16.3 No MM 060 Primary 67 Male 14 9.5 Yes EpithelioidEnucleation 37.0 Yes MM 066 Primary 47 Male 22 9.2 Yes Mixed Enucleation52.5 No MM 070 Primary 62 Male 24 15.6 Yes Epithelioid Enucleation 31.5Yes MM 071 Primary 63 Female N/A 12.5 N/A Spindle Enucleation 46.3 No MM080 Primary 37 Male N/A 11.3 Yes Epithelioid Enucleation 31.5 Yes MM 081Primary 65 Male 18 11.3 Yes Epithelioid Enucleation 28.2 Yes MM 083Primary 43 Male  5 3.7 Yes Epithelioid Enucleation 51.1 Yes MM 087Primary 53 Female 16 5.8 No Epithelioid Enucleation 17.5 Yes MM 090Primary 72 Female 19 14.0 Yes Mixed Enucleation 27.5 No MM 091 Primary64 Male 17 10.2 Yes Mixed Enucleation 26.4 Yes MM 100 Primary 68 Male 1812.3 Yes Epithelioid Enucleation 16.2 No MM 103 Primary 63 Male 15 12.7Yes Mixed Enucleation 33.4 Yes MM 110 Primary 48 Female 15 8.0 NoEpithelioid Enucleation 37.4 No MM 120 Primary 68 Female 20 10.4 YesSpindle Enucleation 26.7 Yes MM 121 Primary 52 Female 17 5.8 Yes SpindleEnucleation 32.3 Yes MM 125 Primary 79 Female 18 3.9 No MixedEnucleation 7. Yes MM 127 Primary 78 Male N/A N/A N/A EpithelioidEnucleation 20.0 No MM 128 Primary 69 Female  8 2.4 No Mixed Enucleation21.1 Yes MM 133 Primary 54 Female 20 15.0 No Epitheliod Enucleation 4.5No MM 134 Primary 57 Female 19 9.1 Yes Mixed Enucleation 6.5 No MM 135Primary 36 Female 20 NA Yes Mixed Enucleation 7.1 No NB 185 Primary 85Male 15 6.9 Yes Epithelioid Brachytherapy 3.8 No NB 191 Primary 60Female  9 2.7 No Spindle Brachytehrapy 3.4 No NB 195 Primary 74 Male 189.0 Yes Mixed Biopsy 3.1 No NB 199 Primary 71 Female 13 8.7 Yes MixedBrachytherapy 1.8 No NB 200 Primary 61 Femlae 18 4.4 Yes SpindleBrachytherapy 6.9 No NB 214 Primary 86 Male 15 4.5 No EpithelioidBrachytherapy 3.4 No MM Metastasis 68 Male 19 15.3 N/A EpitheliodUnknown 4.2 Yes 152M NB 071M Metastasis 51 Male 18 8.6 Yes EpitheliodBrachytherapy 36.5 Yes PVLB Metastasis 43 Female 18 9.0 N/A EpithelioidUnknown 64.4 Yes

Cell Culture:

92.1 (generous gift from Dr. Martine Jager) and Me1290 (generous gift ofDr. Bruce Ksander) human UM cells were grown in RPMI-1640 (Lonza,Walkersville, Md.) supplemented with 10% fetal bovine serum (Invitrogen,Carlsbad, Calif.) and antibiotics. Transfections were performed withHiPerFect (Qiagen, Valencia, Calif.) and Silencer® Select BAP1 (s15820and s15822) or Control #1 siRNA (Ambion, Austin, Tex.). Knockdown ofBAP1 protein levels was confirmed by western blot with antibodies thatrecognize the BAP1 protein (Santa Cruz, Santa Cruz, Calif.) andalpha-tubulin (Sigma-Aldrich, St. Louis, Mo.). Cell morphology data werecollected by digital imaging of phase contrasted cells at 200×magnification. After five days, transfected cells were harvested for RNAand protein analyses.

RNA and Protein Analysis:

All RNA samples were converted to cDNA using the High Capacity cDNAReverse Transcription kit (Applied Biosystems Inc., Foster City, Calif.)and then pre-amplified for 14 cycles with pooled probes and TaqManPre-Amp Master Mix following manufacturer's protocol. Expression of mRNAfor individual genes was quantified using the 7900HT Real-Time PCRSystem with either custom-made primers and iQ SYBRGreen SuperMix(Bio-Rad Laboratories Inc, Hercules, Calif.) for CTNNB1, EDNRB, KIT,SOX10 and UBC (endogenous control) or TaqMan® Gene Expression Assays andGene Expression Master Mix (Applied Biosystems Inc., Foster City,Calif.) for BAP1, CDH1, LTA4H, LMCD1 and ROBO1. The 15-gene prognosticassay for assignment of tumors to class 1 or class 2 was performed asdescribed elsewhere (26). Staining with BAP1 antibody (201C, thegenerous gift of Dr. Richard Baer) was performed on 4 μm sectionsobtained from paraffin-embedded tissue blocks. Statistical significancewas assessed using Student's t-test with Medcalc software version10.4.0.0.

Exome Capture and DNA Sequencing:

gDNA libraries were prepared using the Illumina Pair-End Genomic DNASample Prep Kit (Cat # PE-102-1001) according to the manufacturer'sinstructions. Each paired-end library was enriched for exomic sequenceusing the Roche-Nimblegen SeqCap EZ Exome kit (Cat #5977215001). Thecaptured genomic DNA fragments were sequenced with the Illumina GenomeAnalyzer II (GAIIx) for 76-cycles (one lane per sample).

Sequence Analysis:

Illumina Solexa 76 cycle paired-end sequencing data was received ascompressed raw reads exported from the Illumina software pipeline(≧1.3). Raw reads were parsed into FASTQ format, and the original rawreads were archived. The FASTQ files were aligned to the hg19 version ofthe human reference sequence using bowtie. The Bowtie software (v0.12.3)was compiled with g++ (v4.3.3) using the additional compilation switches“—O3—mtune=amdfam10” with pthreads enabled. Mapped reads were directedto a SAM format file for downstream analysis, and unmapped reads wereexported to a separate file.

Variant bases were extracted with the samtools software (v0.1.7) withthe additional samtools.pl VarFilter switch “-D 1000”, and onlypositions with at least 8 reads and a SNP quality score of at least 20were considered for further analysis. Filtered variants were stored in arelational database table (MySQL v5.0.75). Known SNPs (dbSNP130 on hg19;exact location known, single base changes) and variants found in 8HapMap samples (27) were filtered from our variant lists using databasequeries.

Candidate variants in coding sequence of genes mapping to chromosome 3were identified and manually annotated for amino acid changes. The 30base pairs around coding variants were used to query genomic sequence(hg19) to determine if the sequence mapped to multiple genomiclocations. Regions with multiple identical mappings were removed. Thisincluded the removal of sequences mapping to pseudogenes.

HapMap Variants:

FASTQ files for 8 HapMap individual's exomes (NA19240, NA19129, NA18956,NA18555, NA18517, NA18507, NA12878, NA12156) were downloaded from theNCBI Short Read Archive (3) (accession SRP000910). All reads for eachindividual were aligned to hg19 (see above). Multiple sequencing runswere merged into one SAM formatted file. Variants were extracted withsamtools (see above) and stored in a relational database table.

Sequence Validation:

Oligonucleotide primers were designed from intronic sequences to amplifyall coding sequence of BAP1 with the PCR (Table 5). Genomic DNA of tumorand blood from the same patient were subjected to PCR amplification withroutine approaches. Sanger DNA sequencing was performed with routinemethods to validate variants found with NextGen sequencing, and to queryall tumor and matched normal samples for all coding sequences of BAP1.Oligonucleotide primer sequences are available upon request.

TABLE 5 Sequencing primers PCR product Primers Seq. Exon sizeLocation (hg19) BAP1-e1-3-F2 SEQ ID NO. 15: 1 566 bp chr3:52443441 +AGGCTGCTGCTTTCTGTGAG 3052444006 BAP1-e1-3-R2 SEQ ID NO. 16: 1CGTTGTCTGTGTGTGGGAC BAP1-e4-F SEQ ID NO. 17: 4 261 bp chr3:52442418 -ATGCTGATTGTCTTCTCCCC 52442678 BAP1-e4-R SEQ ID NO. 18: 4CTCCATTTCCACTTCCCAAG BAP1-e5-F SEQ ID NO. 19: 5 255 bp chr3:52441894 -CTTGGGGCTTGCAGTGAG 52442148 BAP1-e5-R SEQ ID NO. 20: 5ATGTGGTAGCATTCCCAGTG BAP1E8L SEQ ID NO. 21: 8 250 bp chr3:52440750 +GGCCTTGCAATTTACAAATCA 52440999 BAP1E8R SEQ ID NO. 22: 8TGTCTTCCTTCCCACTCCTG BAP1-e9-F SEQ ID NO. 23: 9 256 bp chr3:52440207 -GGATATCTGCCTCAACCTGATG 52440462 BAP1-e9-R SEQ ID NO. 24: 9GAAGGGAGGAGGAATGCAG BAP1-e10-F SEQ ID NO. 25: 10 287 bp chr3:52439727 -TTCCTTTAGGTCCTCAGCCC 52440013 BAP1-e10-nest SEQ ID NO. 26: 10This is a nested CTGAGGTCCACAAGAGGTCC primer used for sequencingBAP1-e10-R SEQ ID NO. 27: 10 CAGACATTAGCGGGTGGC BAP1E11L SEQ ID NO. 28:11 227 bp chr3:52439107 + AAGGGTGCTCCCAGCTTAC 52439333 BAP1E11RSEQ ID NO. 29: 11 CCTGTGTTCTTGCCCTGTCT BAP1-e12-F SEQ ID NO. 30: 12270 bp chr3:52438402 - GCTGTGAGTGTCTAGGCTCAG 52438671 BAP1-e12-RSEQ ID NO. 31: 12 AGACTGAGATATTCAGGATGGG BAP1-e14-F SEQ ID NO. 32: 14275 bp chr3:52437098 - CCAAGTGACCACAAAGTGTCC 52437372 BAP1-e14-RSEQ ID NO. 33: 14 AGCTCAGGCCTTACCCTCTG BAP1-e17-F2 SEQ ID NO. 34: 17496 bp chr3:52436103 + CTGAGCACTATGGGGCTGAT 52436598 BAP1-e17-R2SEQ ID NO. 35: 17 TCTTAACTGGAATGCCCTGC BAP1-e13A-F2 SEQ ID NO. 36: 13A567 bp chr3:52437269 + CTGCCTTGGATTGGTCTGAT 52437835 BAP1-e13A-R2SEQ ID NO. 37: 13A CAACACCATCAACGTCTTGG BAP1-e13B-F2 SEQ ID NO. 38: 13B595 bp chr3:52437489 + TGATGACAGGACCCAGATCA 52438083 BAP1-e13B-R2SEQ ID NO. 39: 13B GCTGTCAGAACTTGATGCCA BAP1-e15-16-F SEQ ID NO. 40:15-16 409 bp chr3:52436552 - CTAGCTGCCTATTGCTCGTG 52436960 BAP1-e15-16-RSEQ ID NO. 41: 15-16 GAGGGGAGCTGAAGGACAC BAP1-e6-7-F SEQ ID NO. 42: 6-7412 bp chr3:52441134 - TTTGCCTTCCACCCATAGTC 52441545 BAP1-e6-7-RSEQ ID NO. 43: 6-7 AGCTCCCTAGGAGGTAGGC

DNA Methylation Analysis:

Following bisulfite treatment and amplification of genomic DNA fromregion chr3:52,442,270-52,442,651 (hg19) with bisulfite specificprimers, methylation of this region was evaluated with Sequenom'sMassARRAY Epityper technology in our core facility(hg.wustl.edu/gtcore/methylation.html). Controls for 0% and 100%methylation were also included. Nine class 1 tumors and ten class 2tumors were analyzed.

Molecular Classification:

Gene expression data from custom TaqMan Low-Density Arrays were used todetermine tumor class assignment, as previously described (26). Briefly,molecular class assignments were made by entering the 12 ΔC_(t)values ofeach sample into the machine learning algorithm GIST 2.3 Support VectorMachine (SVM) (bioinformatics.ubc.ca/svm). SVM was trained using a setof 28 well-characterized uveal melanomas of known molecular class andclinical outcome. SVM creates a hyperplane between the training samplegroups (here, class 1 and class 2), then places unknown samples on oneor the other side of the hyperplane based upon their gene expressionprofiles. Confidence is measured by discriminant score, which isinversely proportional to the proximity of the sample to the hyperplane.

Loss of heterozygosity for chromosome 3 was determined using 35 SNPswith minor allele frequencies >0.4 at approximate intervals of 6megabases across the euchromatic regions of chromosome 3 using theMassARRAY system (Sequenom Inc, San Diego, Calif.), as previouslydescribed (25).

Microarray Gene Expression Profiling

Expression data, received as flat files exported from the Illuminasoftware, were analyzed in R (v2.10.1) using Bioconductor packages(Biobase v2.6.1). Non-normalized data were imported into the Renvironment using the beadarray package (v1.14.0). Expression valueswere quantile normalized and log 2 transformed using limma (v3.2.3).Each of three independent siRNA knockdown experiments as well as each ofthree siRNA control experiments was treated as biological replicates.Linear models were fitted to the expression values and expressiondifferences calculated using a contrast comparing the difference inknockdown/control experiments. For each gene log 2 fold change, averageexpression, and moderated t-statistics were calculated for the definedcontrast using the “ebayes” function of the limma package. Nominalp-values were corrected for multiple comparisons using the Benjamini andHochberg false discovery rate method. Heatmaps were generated using theheatmap function of the R base stats package. Quantile normalized datawere filtered down to 29 known discriminating genes plus BAP1. Heatmapcolors were generated using the maPalette function of the marrayBioconductor package (v1.24.0), specifying green as low, red as high,and black as mid color values with 20 colors in the palette.

Example 2 Indirect Methods for Detecting BAP1 Loss

BAP1 loss leads to biochemical changes in the cell, such as histone H2Aubiquitination, that may be easier to detect and monitor than directBAP1 activity.

BAP1 stable knockdown cells were produced using lentiviral vectorsexpressing a short hairpin RNA (shRNA) against BAP1 (FIG. 8). Bothtransient and stable knockdown of BAP1 lead to increased ubiquitinationof histone H2A (FIG. 9). Thus, the measurement of histone H2Aubiquitination levels could be used as a surrogate indicator of BAP1loss.

Stable knockdown of BAP1 also leads to a decrease in the RNA levels ofmelanocyte differentiation genes (FIG. 10). Transient knockdown of BAP1leads to a decrease in proliferation (FIG. 11) as measured using a BrdUassay. In addition, loss of BAP1 in culture leads to decreased cellmotility (FIG. 12) and a decreased growth in soft agar (FIG. 13). On theother hand, loss of BAP1 leads to an increased ability to grow inclonegenic assays (FIG. 14) and increased migration towards a serumattractant (FIG. 15).

Example 3 Loss of BAP1 and Tumor Behavior in Mouse

Uveal melanoma cells stably knocked down for BAP1 using lentiviralexpression of shRNA against BAP1 were implanted into mouse flank. Cellsdeficient for BAP1 grew less rapidly in the mouse flank compared tocontrol cells infected with lentiviral vector expression shRNA againstGFP (FIG. 16). After injection into the tail vein of mice, knockdownBAP1 cells exhibited decreased tumor growth (FIG. 17). These findings,coupled with the cell culture experiments above, indicate that the majoreffect of BAP1 loss in uveal melanoma is not increased proliferation,migration, motility or tumorigenicity upon flank injection.

Example 4 BAP1 Mutations in Cutaneous Melanoma

BAP1 mutations may also be analyzed in cutaneous melanoma tumors asdescribed in the examples and materials and methods above. Cutaneousmelanoma tumors analyzed may be atypical moles (Dysplastic Nevus), basalcell carcinomas, blue nevi, cherry hemangiomas, dermatofibromas, halonevi, keloid and hypertrophic scars, keratoacanthomas, lentigos,metastatic carcinomas of the skin, nevi of ota and ito, melanocyticnevi, seborrheic keratosis, spitz nevi, squamous cell carcinomas, andvitiligos.

Cutaneous melanoma samples and matching normal DNA from peripheraltissue may be analyzed for inactivating mutations in BAP1 using exomecapture followed by massively parallel sequencing. Sanger re-sequencingof all BAP1 exons may also be used to further investigate BAP1mutations. Normal DNA from patients with cutaneous melanoma may beanalyzed to determine if BAP1 mutations are somatic or germline inorigin. Germline alterations in BAP1 may predispose to cutaneousmelanoma.

Mutation status of other genes may also be analyzed in the cutaneousmelanoma samples. For example, GNAQ, BRAF, KIT or NRAS mutation statusmay be determined, and compared to the results obtained for uvealmelanoma samples described above.

BAP1 mRNA levels may be analyzed using quantitative RT-PCR. If BAP1 mRNAlevels are lower in cutaneous melanoma samples than in normal samples,DNA methylation of the BAP1 locus may be analyzed to determine if thelower mRNA levels may be explained by DNA methylation. BAP1 proteinlevels in various tumor and normal samples may also be analyzed usingimmunofluorescence.

BAP1 may be knocked down in cell culture using RNAi. BAP1 mRNA andprotein expression levels, cell morphology, and gene expressionprofiling using microarrays may be used to characterize cell culturesafter knock down of BAP1 expression.

Example 5 BAP1 Mutations in the Germline

BAP1 mutations may be detected in germline DNA as a means of detectionof affected family members in hereditary syndromes. Germline DNA may beany normal patient DNA such as DNA extracted from peripheral bloodlymphocytes or buccal swabs. Standard Sanger sequencing may be used asdescribed in Example 1 above.

For instance, FIG. 18 illustrates a family with stomach cancer, bonecancer, breast cancer, bladder cancer, uveal melanoma, and cutaneousmelanoma. The individuals labeled FUM1-01, FUM1-02, FUM1-03, and FUM1-04were positive for germline BAP1 mutations. These data support theconclusion that germline BAP1 mutations may be used to detect affectedfamily members in hereditary cancers and/or syndromes.

Example 6 BAP1 as a Marker of Circulating Tumors

BAP1 mutations may be detected in peripheral blood as a marker ofcirculating tumor cells. This may be performed using targeted captureand deep sequencing of BAP1 in blood samples from patients. Targetedcapture may be used in combination with NexGen sequencing to provide avery powerful approach for rapidly sequencing genomic regions ofinterest. The Agilent SureSelect enrichment system is one such methodthat allows enrichment for genomic regions from a sample of total humangenomic DNA. The Agilent system also supports multiplexing of samples inthe sequencing reaction, reducing the overall cost of the procedure.

A 1-2 Mb genomic region harboring BAP1 may be captured. This may allowdetection of deletions of several exons or the entire gene, as well asthe smaller mutations identified in the examples above. Targeted capturewith Agilent's SureSelect system starts with querying their eArray website for a region of interest. This is designed to identify anoverlapping set of oligonucleotides (120 mers) over a particular region,but without regions containing repeat (which confound the selectionprocedure). Agilent synthesizes biotinylated cRNA oligonucleotides andprovides them in solution (the probe). 1-3 mg of genomic DNA (thedriver) may then be sheared to ˜200 bp, end-repaired, A-tailed andligated to adaptors for Illumina paired-end sequencing. Libraries may beamplified for 6-8 cycles to produce at least 500 ng of product. Theproduct may be hybridized to the oligonucleotide baits to enrich fortargeted regions then the resultant hybrids may be captured ontostreptavidin-labeled magnetic beads. This may be followed by washing anddigestion of the RNA bait. Resultant selection products may be subjectedto PCR for 12-14 cycles. At this stage, unique oligonucleotideidentifiers may be incorporated into the selected DNAs and theirconcentrations are determined. These are then adjusted it to a finalconcentration of 15 pM for sequencing. In this way multiple samples maybe loaded onto one flow cell lane on the Sequencer. Currently, 12samples may be run in a single lane of an Illumina HiSeq2000. Illuminaand Nimblegen are also developing similar technologies that could beused for targeted capture. This technology was originally developed byDr. Michael Lovett (Bashiardes et al. 2005), and instead ofoligonucleotides, bacterial artificial chromosomes (BACs) were used asprobes. Hence, there are a variety of ways of identifying the genomictarget of interest.

Sequences obtained from targeted capture may be analyzed in a similarmanner to those obtained from exome-capture and as described elsewhere.

This may potentially be used for (1) non-invasive determination ofpatients with class 2 high risk uveal melanomas, (2) assessment ofcirculating tumor burden for uveal, cutaneous or other BAP1 mutantcancer, and (3) to monitor response to therapy.

REFERENCES

-   1. Landreville S, Agapova O A, Harbour J W. Future Oncol. 2008;    4:629.-   2. Onken M D, Worley L A, Tuscan M D, Harbour J W. J Mol Diagn.    2010; 12:461.-   3. Finger P T. Arch Pathol Lab Med. 2009; 133:1197.-   4. Van Raamsdonk C D, et al. Nature. 2009; 457:599.-   5. Onken M D, et al. Invest Ophthalmol Vis Sci. 2008; 49:5230.-   6. Bauer J, et al. Br J Cancer. 2009; 101:813.-   7. Worley L A, et al. Clin Cancer Res. 2007; 13:1466.-   8. Bashiardes S, et al. Nat Methods. 2005; 2:63.-   9. Ng S B, et al. Nat Genet. 2010; 42:30.-   10. Jensen D E, et al. Oncogene. 1998; 16:1097.-   11. Misaghi S, et al. Mol Cell Biol. 2009; 29:2181.-   12. Nishikawa H, et al. Cancer Res. 2009; 69:111.-   13. Machida Y J, Machida Y, Vashisht A A, Wohlschlegel J A, Dutta A.    J Biol Chem. 2009; 284:34179.-   14. Tyagi S, Chabes A L, Wysocka J, Herr W. Mol Cell. 2007; 27:107.-   15. Gaytan de Ayala Alonso A, et al. Genetics. 2007; 176:2099.-   16. Scheuermann J C, et al. Nature. 2010; 465:243.-   17. Wood L D, et al. Science. 2007; 318:1108.-   18. Onken M D, et al. Cancer Res. 2006; 66:4602.-   19. Onken M D, Worley L A, Ehlers J P, Harbour J W. Cancer Res.    2004; 64:7205.-   20. D. Lang et al., Nature 433, 884 (Feb. 24, 2005).-   21. L. A. Garraway et al., Nature 436, 117 (Jul. 7, 2005).-   22. T. J. Hemesath, E. R. Price, C. Takemoto, T. Badalian, D. E.    Fisher, Nature 391, 298 (Jan. 15, 1998).-   23. Misaghi S, et al. Journal of Biological Chemistry. 2005;    280:1512.-   24. Wang Y, et al. Nucleic Acids Res. 2007; 35:D298.-   25. M. D. Onken et al., Clin Cancer Res 13, 2923 (2007).-   26. M. D. Onken, L. A. Worley, M. D. Tuscan, J. W. Harbour, J Mol    Diagn 12, 461 (2010).-   27. S. B. Ng et al., Nature 461, 272 (2009).

1.-15. (canceled)
 16. A method for determining the risk of melanoma metastasis in a subject, the method comprising: (a) analyzing BAP1 nucleic acid from a cell in a sample obtained from a subject, (b) detecting the presence of a truncating mutation in the BAP1 nucleic acid, wherein the mutation is selected from the group consisting of: i. a nonsense mutation selected from the group consisting of Q36X, W196X and Q253X of BAP1; ii. an insertion or deletion mutation in exon 2, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 17 of BAP1; and iii. a splice acceptor mutation in exon 16 of BAP1; and (c) identifying the subject as having an increased risk for metastasis when a mutation is detected.
 17. The method of claim 16, wherein the melanoma is uveal melanoma.
 18. The method of claim 16, wherein the sample is a tumor sample.
 19. The method of claim 18, wherein the sample is collected from a primary tumor or from a circulating tumor cell.
 20. The method of claim 19, wherein the circulating tumor cell is collected from a bodily fluid.
 21. A method for prognosing melanoma in a subject, the method comprising: (a) analyzing BAP1 nucleic acid from a cell in a sample obtained from a subject, (b) detecting the presence of a truncating mutation in the BAP1 nucleic acid, wherein the mutation is selected from the group consisting of: i. a nonsense mutation selected from the group consisting of Q36X, W196X and Q253X of BAP1; and ii. an insertion or deletion mutation in exon 2, 4, 5, 6, 7, 8 or 9 of BAP1; and (c) identifying the subject as having poor prognosis when a mutation is detected.
 22. The method of claim 21, wherein the melanoma is uveal melanoma.
 23. The method of claim 21, wherein the sample is a tumor sample.
 24. The method of claim 23, wherein the sample is collected from a primary tumor or from a circulating tumor cell.
 25. The method of claim 24, wherein the circulating tumor cell is collected from a bodily fluid. 